HUWE1 deficiency enhances characteristics of CSCs in CRC cells

To examine the role of HUWE1 deficiency in promoting stemness in CRC, we suppressed HUWE1 expression in DLD1, HT29, and HCT116 cells using lentivirus-delivered shRNAs. Compared to control cells, HUWE1-deficient CRC cells exhibited increased sphere formation and colony formation capacity (Fig. 1A, B). FACS analysis further revealed a higher proportion of CD24/CD133 cells in HUWE1-deficient CRC cells (Fig. 1C, Supplementary Fig. 1A). CD24 and CD133 are well-known putative markers of the cancer stem cell population in CRC and are associated with aggressive tumor behavior [22,23,24]. Therefore, these results supported the notion that HUWE1-deficient CRC cells represent a cancer stem cell population. We also detected that expression of LGR5 and pluripotency transcription factors involved in stem cell maintenance was upregulated in HUWE1-deficient CRC cells (Fig. 1D, Supplementary Fig. 1B). Furthermore, HUWE1-deficient CRC cells displayed increased IC50 values for oxaliplatin, 5-fluorouracil (5FU), and doxorubicin, indicating enhanced chemoresistance (Fig. 1E). Consistently, these cells showed a significant reduction in apoptosis compared to control cells (Fig. 1F, Supplementary Fig. 1C). We also observed a greater shift of the PI+ population in the control group following doxorubicin treatment. This shift reflects increased intracellular accumulation of doxorubicin -- which exhibits similar fluorescence similar to PI -- rather than an actual increase in cell death. These findings suggest that HUWE1 loss enhances stemness and confers drug resistance in CRC cells.

To evaluate whether HUWE1 depletion affects cell growth in CRC cells, we performed a CCK-8 assay and found that suppression of HUWE1 significantly increased cell proliferation (Fig. 2A). Consistently, the number of EdU-positive cells was notably high in HUWE1-deficient CRC cells compared to the control cells (Fig. 2B). Next, we investigated the effect of HUWE1 depletion on the migration and invasion of CRC cells. HUWE1-deficient CRC cells exhibited faster wound closure, indicating an increased migration rate (Fig. 2C). Similarly, a transwell invasion assay revealed a higher number of invaded cells in HUWE1-deficient CRC cells compared to controls (Fig. 2D). We further examined the role of HUWE1 depletion in regulating epithelial-mesenchymal transition (EMT) markers using immunofluorescence analysis. Compared to control cells, HUWE1-deficient CRC cells displayed decreased E-cadherin expression and increased levels of N-cadherin and Vimentin (Fig. 2E). Consistent with these findings, western blot analysis revealed reduced E-cadherin expression alongside increased expression of N-cadherin, Vimentin, Snail, and Slug in HUWE1-deficient CRC cells (Fig. 2F, Supplementary Fig. 2A). In addition, cellular morphological analysis using Phalloidin-iFluor 647 Reagent revealed that control CRC cells exhibited a typical cuboidal morphology, whereas HUWE1-deficient CRC cells displayed a more elongated and mesenchymal-like phenotype (Supplementary Fig. 2B). These results suggest that loss of HUWE1 promotes cell proliferation, migration, invasion, and EMT progression in CRC cells.

To investigate whether HUWE1 depletion affects the regulation of Wnt/β-catenin signaling, we analyzed β-catenin expression levels in HUWE1-deficient CRC cells. Immunofluorescence analysis revealed a significant accumulation of β-catenin in both the nucleus and cytoplasm of HUWE1-deficient CRC cells (Fig. 3A). Consistently, western blot analysis demonstrated increased expression of Wnt/β-catenin downstream target genes, including c-Myc and Cyclin D1, in HUWE1-deficient CRC cells (Fig. 3B, Supplementary Fig. 3A).

To determine whether HUWE1 depletion impacts β-catenin ubiquitination, we performed a ubiquitination assay. HUWE1-deficient CRC cells were co-transfected with plasmids containing Flag-β-catenin and HA-Ub, followed by co-immunoprecipitation (Co-IP) to detect ubiquitinated β-catenin. The results showed a reduction in β-catenin ubiquitination in HUWE1-deficient DLD1 and HT29 cells compared to control cells (Fig. 3C). However, HUWE1-deficient HCT116 cells, which possess wild-type APC, exhibited no significant changes in β-catenin ubiquitination levels. Previous studies have shown that HUWE1 loss results in excessive β-catenin accumulation specifically in the absence of functional APC [15]. Given the high prevalence of APC mutations in CRC (Supplementary Fig. 3B), we examined whether the HUWE1 depletion similarly impacts β-catenin accumulation in other types of cancer cells with the wild-type APC. In DLD1 cells with truncated APC, HUWE1 suppression resulted in a substantial accumulation of β-catenin, a phenomenon not observed in other cancer cells with intact APC (Fig. 3D, Supplementary Fig. 3C). Furthermore, in RKO cells, which have an intact Wnt/β-catenin signaling pathway, dual inhibition of HUWE1 and APC led to greater β-catenin accumulation compared to APC inhibition alone (Supplementary Fig. 3D). Collectively, these findings indicate that HUWE1 facilitates β-catenin degradation, particularly in the context of APC dysfunction, highlighting its role as a key regulator of Wnt/β-catenin signaling in CRC.

To confirm the specificity of HUWE1 knockdown and perform rescue experiments, we generated a HUWE1 construct (resHUWE1) resistant to the target nucleotide sequence of the HUWE1 shRNA (Supplementary Fig. 3E). Using the construct, we assessed the degradation of β-catenin upon overexpression of resHUWE1 or full-length APC in HUWE1-deficient CRC cells. In HUWE1-deficient DLD1 and HT29 cells, overexpression of resHUWE1 or full-length APC significantly increased β-catenin ubiquitination (Fig. 3E). However, in HUWE1-deficient HCT116 cells (which harbor a serine 45 deletion in β-catenin), no notable differences in β-catenin ubiquitination were observed.

Interestingly, overexpression of resHUWE1 reduced the expression of endogenous β-catenin and its downstream target proteins (c-Myc and Cyclin D1) across HUWE1-deficient DLD1, HT29, and HCT116 cells (Fig. 3F, Supplementary Fig. 3F). In contrast, overexpression of full-length APC failed to reduce β-catenin levels and downstream target protein expression in HUWE1-deficient HCT116 cells (Fig. 3F, Supplementary Fig. 3F). Moreover, overexpression of the catalytically inactive resHUWE1 C4341S mutant also failed to decrease β-catenin and its downstream target proteins, clearly demonstrating that the degradation of β-catenin is dependent on the E3 ligase activity of HUWE1 (Supplementary Fig. 4). These results suggest that HUWE1 specifically regulates β-catenin degradation and Wnt/β-catenin signaling under conditions of β-catenin destruction complex dysfunction, particularly when APC function is impaired or β-catenin mutations disrupt the normal degradation pathway.

The results described above suggest that HUWE1 plays a role in β-catenin degradation, similarly to the β-catenin destruction complex. However, it is unclear whether HUWE1 directly degrades β-catenin when the β-catenin destruction complex is functional. To address this, we overexpressed full-length APC in CRC cells and evaluated the interaction between HUWE1 and β-catenin using Co-IP. Overexpression of full-length APC in DLD1 and HT29 cells significantly reduced the interaction between HUWE1 and β-catenin (Fig. 4A). Interestingly, overexpression of full-length APC in DLD1 and HT29 cells displayed the interaction between HUWE1 and upstream Wnt/β-catenin signaling proteins, such as DVL1 and PPDPF, whereas no such interaction was observed in HCT116 cells (Fig. 4B, C). These findings were further validated by proximity ligation assay (PLA). We detected amplified PLA signals in DLD1, HT29, and HCT116 cells labeled with HUWE1 and β-catenin primary antibodies, but no signals were observed in cells labeled with HUWE1 and Dvl or HUWE1 and PPDPF primary antibodies (Fig. 4D). However, overexpression of full-length APC reduced HUWE1-β-catenin PLA signals only in DLD1 and HT29 cells, but not in HCT116 cells, while signals corresponding to HUWE1-DVL1 and HUWE1-PPDPF interactions became apparent (Fig. 4E). Furthermore, in RKO cells, inhibition of APC led to an increased interaction between HUWE1 and β-catenin, while the interactions between HUWE1 and Dvl, as well as HUWE1 and PPDPF, were decreased (Supplementary Fig. 5A-D). These results suggest that in the Wnt-on hyperactivation state, HUWE1 primarily facilitates β-catenin degradation under conditions of β-catenin destruction complex dysfunction caused by mutations in APC (as in DLD1 and HT29 cells) or β-catenin (as in HCT116 cells). On the other hand, in the conditions where the β-catenin destruction complex remains functional (as in RKO cells), HUWE1 interacts with Dvl or PPDPF to fine-tune Wnt/β-catenin signaling (Fig. 4F).

Next, we investigated whether the overexpression of resHUWE1 could reverse the enhanced cell proliferation, migration, invasion, and stemness induced by HUWE1 depletion in CRC cells. Overexpression of resHUWE1 significantly reduced the sphere formation efficiency and colony formation capacity in HUWE1-deficient DLD1 and HCT116 cells (Fig. 5A, B). In addition, resHUWE1 overexpression inhibited cell proliferation in HUWE1-deficient DLD1 and HCT116 cells (Fig. 5C). Consistently, we observed a decrease in EdU-positive cells upon resHUWE1 overexpression, indicating reduced DNA synthesis and proliferation (Fig. 5D). Furthermore, resHUWE1 overexpression reduced both cell migration and invasion rates in HUWE1-deficient DLD1 and HCT116 cells (Fig. 5E, F). Western blot analysis revealed that resHUWE1 overexpression reduces the expression of stemness-associated markers, including LGR5, OCT4, SOX2, and NANOG, in HUWE1-deficient DLD1 and HCT116 cells (Fig. 5G, Supplementary Fig. 6A). Overexpression of resHUWE1 also led to an increase in the expression of E-cadherin and decreased expression of N-cadherin, Vimentin, Snail, and Slug in HUWE1-deficient CRC cells (Fig. 5H, Supplementary Fig. 6B). Interestingly, while full-length APC overexpression effectively reduced cell proliferation, migration, invasion, and stemness in HUWE1-deficient DLD1 cells, it failed to produce the same effect in HUWE1-deficient HCT116 cells. Moreover, in HUWE1-deficient DLD1 cells, these effects were increased when both resHUWE1 and APC were overexpressed simultaneously compared to APC overexpression alone. We also demonstrated that overexpression of wild-type resHUWE1 reduced the expression of stemness-associated markers in HUWE1-deficient DLD1 and HCT116 cells, whereas the catalytically inactive resHUWE1 C4341S mutant showed no such effect (Supplementary Fig. 7). These findings collectively indicate that HUWE1 regulates Wnt/β-catenin signaling in CRC in coordination with the β-catenin destruction complex, playing a crucial role in controlling cell proliferation, migration, invasion, and stemness.

Next, we investigated whether mitochondrial biogenesis is upregulated by HUWE1 depletion in CRC cells. We observed an increase in the expression of key transcriptional regulators involved in mitochondrial biogenesis, such as SIRT1, PGC1α, NRF2, and TFAM, in HUWE1-deficient CRC cells (Fig. 6A, Supplementary Fig. 8A). Additionally, the expression of mitochondrial electron transfer chain (ETC) complex subunit proteins, including NDUFB8, SDHB, UQCRC2, COXIV, and ATP5A1, was also elevated in HUWE1-deficient CRC cells (Fig. 6B, Supplementary Fig. 8B). To further validate these results, we analyzed the TCGA CRC dataset and found that HUWE1 expression was negatively correlated with the expression levels of most mitochondrial proteins (Fig. 6C). Moreover, we observed that the ETC activity and ATP production were significantly enhanced in HUWE1-deficient CRC cells compared to the control cells (Fig. 6D-G). These results suggest that depletion of HUWE1 promotes mitochondrial biogenesis, inducing ETC activity, and consequently increasing ATP production in CRC cells.

Additionally, we observed elevated expression of ABC transporter proteins in HUWE1-deficient CRC cells (Fig. 6H, Supplementary Fig. 8C). To assess the impact of HUWE1 depletion on drug efflux effects, we measured the release of doxorubicin over a 24 h period. While control cells retained doxorubicin, it was nearly absent in the HUWE1-deficient CRC cells (Fig. 6I, Supplementary Fig. 8D). In addition, pharmacological inhibition of mitochondrial ATP production with oligomycin increased drug sensitivity in HUWE1-deficient CRC cells (Supplementary Fig. 9A, B). Similarly, inhibiting ABC transporter activity with tariquidar also enhanced drug sensitivity (Supplementary Fig. 9C). Collectively, these findings suggest that loss of HUWE1 in CRC cells leads to increased ATP production, which likely supports the overexpressed ABC transporter proteins, contributing to drug resistance.

To investigate whether overexpression of resHUWE1 could modulate mitochondrial biogenesis enhanced by HUWE1 depletion in CRC cells. We found that resHUWE1 overexpression significantly reduced the expression of proteins involved in mitochondrial biogenesis and ETC complex subunit in both HUWE1-deficient DLD1 and HCT116 cells, whereas overexpression of full-length APC only slightly decreased the expression of these proteins in HUWE1-deficient DLD1 cells (Fig. 7A, B, Supplementary Fig. 10A, B). Moreover, resHUWE1 overexpression led to a decrease in ETC activity and ATP production in both HUWE1-deficient DLD1 and HCT116 cells, whereas overexpression of full-length APC had a mild or no effect on these in HUWE1-deficient DLD1 or HCT116 cells, respectively (Fig. 7C-F). These results indicate that HUWE1 plays a crucial role in regulating mitochondrial biogenesis in CRC cells.

To determine whether drug resistance induced by HUWE1 depletion in CRC cells could be reversed upon HUWE1 restoration, we overexpressed resHUWE1 in HUWE1-deficient DLD1 and HCT116 cells and assessed their drug sensitivity. We observed a decrease in IC50 values for oxaliplatin, 5FU, and doxorubicin in both HUWE1-deficient DLD1 and HCT116 cells upon resHUWE1 overexpression (Fig. 7G). Furthermore, resHUWE1 overexpression significantly enhanced apoptosis in both HUWE1-deficient DLD1 and HCT116 cells (Fig. 7H, Supplementary Fig. 10C). In contrast, full-length APC overexpression increased drug sensitivity and apoptosis only in HUWE1-deficient DLD1 cells, and the effect was less pronounced than with resHUWE1 overexpression. Taken together, these results suggest that HUWE1 contributes to increased drug sensitivity by regulating not only Wnt/β-catenin signaling but also mitochondrial biogenesis in CRC cells.